RS20140200A1 - POLYMER NANOCOMPOSITES WITH INCORPORATED SILVER AND Honey Nanoparticles - Google Patents

Abstract

Polymeric nanocomposites containing honey and silver nanoparticles are described where the nanocomposites are in the form of a colloidal solution or hydrogel based on alginate or poly-vinyl alcohol. These nanocomposites, which are in the form of particles, microparticles, fibers, microfibers, plates or films, are gelled forms of polymer solutions such as sodium alginate or poly-vinyl alcohol wherein the solutions comprise 50-95% by weight of colloidal sodium alginate solution with silver nanoparticles , and 5-50% by weight of honey, ie. 70-95% by weight of colloidal solution of poly-vinyl alcohol with silver nanoparticles and 5-30% by weight of honey. Also disclosed is a process for the preparation of polymer nanocomposites with incorporated copper and silver nanoparticles.

Description

POLYMER NANOCOMPOSITES WITH INCORPORATED SILVER AND HONEY NANOPARTICLES

This invention relates to dressings for biomedical applications, and more specifically to dressings that are polymer nanocomposites with incorporated nanoparticles of silver and honey, where these nanocomposites are in the form of a colloidal solution or hydrogel on a base of alginate or polyvinyl alcohol.

Wounds, whether chronic or acute, represent a significant problem in medical practice and hence there are various approaches to wound treatment. One way of treatment is the application of alginate-based dressings that effectively regulate wound moisture leading to faster granulation and re-epithelialization of damaged tissue, as described by W. Paul, CP. Sharma. Trends Biomater Artif Organs 18 (2004) p. 18. and D. Queen, H. Orsted, H. Sanada, G. Sussman. Int Wound J 1 (2004) p. 59., which is why such dressings have found wide application for the treatment of various wounds. However, these dressings are not the most effective in treating infected wounds because they do not have antimicrobial properties. Additionally, one of the problems with wounds, especially chronic wounds, is an elevated pH value ranging from 7.15 to 8.9 as shown by G. Gethin. Wounds UK 3 (2007) p. 52., while the pH value of normal skin is between 4 and 6 depending on anatomical location and age (LA. Schneider, A. Korber, S. Grabbe, J. Dissemond. Arch Dermatol Res 298 (2007) p. 413.). For this reason, it is desirable that the pH value of preparations for the treatment of wounds be slightly acidic, because it has been shown that wounds, whether acute or chronic, heal more slowly if their pH is elevated, i.e. alkaline, than when it is close to neutral. The acidity of the wound accelerates its healing because the percentage of oxygen increases by its release from hemoglobin from capillaries (M. Rendel, C. Mayer, W. Vveninger, E. Tschachler. Br J Dermatol 145 (2001) p. 3.) and proteinase activity is suppressed, which slows down or prevents wound healing because it destroys growth factors, protein fibers and fibronectin in the wound matrix, which, in turn, is necessary for the activation of fibroblasts and their migration (I. Rushton. Nurs Stand 21 (2007) p. 68.).

The functionality of alginate dressings can be improved by the addition of honey due to a number of beneficial properties that honey has, such as antibacterial activity (R.A. Cooper, E. Halas, P.C. Molan. J Bum Care Rehabil 23 (2002) p. 366.; and P.E. Lusbv, A.L. Coombes, J.M. Wilkinson. Arch Med Res 36 (2005) p. 464.), anti-inflammatory effects (P. C. Molan. Buli. Eur. Tissue Rep. Soc. 9 (2002) p. 5., and US RE42,755 E), stimulation of angiogenesis (K. Rossiter, A. J. Cooper, D. Voegeli, B. A. Lwaleed. J VWound Care 19 (2010) p.

2.) and accelerating wound healing (S.S. Gupta, O. Singh, P.S. Bhagel, S. Moses, S. Shukla, R.K. Mathur. J Cutan Aesthet Surg 4 (2011) p. 183.). Several commercial products based on alginate and honey have been developed, in most cases alginate fibers are dipped in honey for medical use, such as Vivamel (Tosama, Slovenia), Algivon (Bamford, New Zealand) and MediHonev<®>Calcium Alginate Dressing (Đerma Sciences, USA). Also, US RE 42,755 E describes the process of obtaining alginate dressings with honey by mixing sodium alginate powder and honey at a relatively high temperature (60°C), whereby the alginate dissolves and absorbs water from the honey, which leads to the formation of a gel. Gels prepared in this way are in the form of sheets or plates or can be poured into molds, but they are easily soluble, so they can be additionally gelled by adding polyvalent cations. The disadvantage of this invention is the relatively high temperature at which the alginate is gelled, because honey is known to lose its medicinal properties at temperatures higher than 41°C. In general, honey lowers the pH of the dressing so that G.T. Gethin, S. Cowman, R.M. Conroy. Int Wound J 5 (2008) p. 185., showed that when chronic non-healing wounds are treated with poultices with honey whose pH = 4, the pH of the wound decreases from 7.72 to 7.26 after two weeks of treatment, which leads to a reduction in the size of the wound. According to these authors, a decrease in pH by 0.1 unit leads to a decrease in wound size by 8.1%. However, in addition to the significant positive properties of alginate dressings with honey in the treatment of wounds, one negative side has been observed in practice and consists in the fact that the replacement of these dressings is somewhat painful because the dressing sticks to the wound due to the presence of honey.

On the other hand, effective antimicrobial activity of alginate wound dressings is achieved by adding silver or silver-based compounds, because the strong antimicrobial activity of this metal is known, as well as the broad inhibitory and biocidal spectrum of silver against various microorganisms (R.M. Slavson, M.l Van Dyke, H. Lee, J.T. Trevors, Plasmid. 27 (1992) p. 72.; R. Bhattacharya, P. Mukherjee. Adv. Drug Delivery Rev. 60 (2008) p. 1289. and I. Dottori, S. Mattioli. For these reasons, commercial coatings based on alginate have been developed with the addition of silver in ionic form, as well as several products with silver nanoparticles on the surface of the polymer, such as Acticoat (Smith & Nephew).

To date, a large number of in vitro and in vivo studies have been conducted investigating how silver or honey dressings affect wound healing (M. Subrahmanyam. Burns 24 (1998) p. 157; and B.K.H.L. Boekema, L. Pool, M.M.VV. Ulrich. Burns 39 (2012) p. 754.). All studies have shown that compresses with silver suppress infection more effectively than compresses with honey, because silver is a broad-spectrum antimicrobial agent. On the other hand, poultices with honey significantly accelerate the re-epithelialization of damaged tissue, unlike poultices with silver, which delay it because they stimulate pro-inflammatory cytokines.

In the application of silver as an antimicrobial agent, nanoparticles are thought to be more potent than silver ions, with smaller nanoparticles having greater activity (V.K.Sharma, R.A. Yngard, Y, Lin. Adv. Colloid Interface Sci 145 (2009) p. 83). Silver nanoparticles are obtained by various methods including chemical and electrochemical synthesis in polymer solutions. During the chemical synthesis, it was shown that small, uniform nanoparticles are obtained when silver reduction takes place in an alkaline environment (N. Leopold, B. Lendl, J. Phys. Chem. B 107 (2003), p. 5723). Sodium hydroxide is most often used to raise the pH value of the reaction mixture, whereby as the pH value increases, the reduction of silver accelerates and the diameter of the nanoparticles decreases, so that uniform, spherical and small nanoparticles are obtained at pH values of 10.5 to 11. Likewise, during the electrochemical synthesis of silver nanoparticles in aqueous solutions of sodium alginate (B. Obradović et al., RS patent application P-2010/0499), obtained colloidal solutions have high pH values in the range from 9 to 11. For this reason, chemically and electrochemically synthesized colloidal solutions with silver nanoparticles have a high pH value that needs to be lowered in case of application for wound treatment, but so that the silver nanoparticles remain stable. When the pH value of the colloidal solution containing silver nanoparticles (pH around 11) is lowered with an organic acid, such as ascorbic acid, the silver nanoparticles become unstable after 10 days (J. Stojkovska et al., Rane 4, 2013).

Honey was also used for the synthesis of silver nanoparticles both as a reducing agent and as a stabilizer, with glucose serving to reduce the silver and honey proteins to stabilize the nanoparticles. The synthesis takes place in the presence of sodium hydroxide at pH values in the range of 6.5 to 8.5 (D. Philip. Spectrochim Acta Part A 75 (2010) p. 1078.). This procedure was investigated for the development of an environmentally acceptable procedure for the synthesis of silver nanoparticles at a honey concentration of about 8.5%, which is significantly lower than the concentration required for honey to exhibit antimicrobial activity, which is at least 30% (C. Basualdo, V. Sgroy, M. S. Finola, J. M. Marioli. Vet Microbiol 124 (2007) p. 375.).

From the previously described state of the art, it follows that the problem of high pH value of polymer colloidal solutions with silver nanoparticles obtained by certain procedures of electrochemical or chemical synthesis remains unresolved, and which colloidal solutions are intended for use in the treatment of wounds caused by any means. Also, the problem of providing dressings for wounds that would contain honey in them, and not only on their surface, and that the concentration of certain components of honey in the dressings is sufficient for honey to show its activity in improved wound healing, remains unsolved.

The essence of this invention is new products based on colloidal solutions or polymer hydrogels that contain silver nanoparticles and honey, where the colloidal solutions and hydrogels are based on alginate and poly-vinyl alcohol (PVA). At the same time, adding honey to the solutions of the mentioned polymers with silver nanoparticles achieves (i) a lowering of the pH value of the solution, which enables the application of these solutions in the treatment of wounds, (ii) the stabilization of silver nanoparticles in a slightly acidic environment, which enables prolonged use of the solution, and (iii) a synergistic effect of alginate or PVA, honey and silver nanoparticles in the wound healing solution. In addition, from the obtained solutions of alginate or PVA, nanocomposite hydrogels with incorporated nanoparticles of silver and honey were produced, in different forms such as films, plates, microparticles, microfibers, which can be used in wound treatment, both in wet and dry form.

The advantage of the nanocomposite hydrogels according to the present invention is that they enable the production of wound dressings, which show antimicrobial activity and the positive effect that honey has on wound healing. In addition, since the honey is contained within the alginate hydrogel, the dressing is not sticky, so its replacement during the treatment, when necessary, is less traumatic for the treated individual.

The present invention provides stable colloidal solution mixtures with silver nanoparticles and honey with pH values suitable for use in wound treatment. Mixtures are obtained by mixing honey and a colloidal solution of polymers, such as e.g. sodium alginate or poly-vinyl alcohol, with contained silver nanoparticles, at room temperature in a certain ratio, as shown in the examples, whereby the pH value is lowered and the nanoparticles remain stable. Depending on the initial pH values of the colloidal solution and honey, the effects of reducing the pH value are observed even with the addition of the smallest amounts of honey, but the reduction to the desired range of pH values between 4 and 8, and more specifically between 5 and 6, is obtained by adding honey of at least about 5 mass. %. The pH value of the mixture decreases with an increase in the mass fraction of honey tending to the pH value of pure honey, but at pH values below 5 the stability of nanoparticles generally decreases.

Polymer hydrogels, such as sodium alginate or PVA, with incorporated silver nanoparticles and honey, according to this invention, are obtained by gelling the obtained colloidal solution with honey in solutions of salts of multivalent cations, i.e. gelling agent. Nanocomposite hydrogels in the form of spherical particles, microparticles, then fibers and microfibers, according to this invention, are obtained by extruding a colloidal solution into a gelling solution containing multivalent ions, i.e. a gelling agent, while nanocomposite hydrogels in the form of plates and films are obtained by pouring the colloidal solution into molds and then drying and/or gelling in the gelling solution.

The products provided by this invention and the processes for obtaining them are described below through the following examples.

Example 1. Refusal of a stable mixture of alginate, honey and silver nanoparticles

Sodium-alginate colloidal solution with electrochemically synthesized silver nanoparticles (pH~ll containing 2 wt.% alginate, 1 mM silver nanoparticles) was mixed with acacia honey in a mass ratio of 3:2. After adding honey, the pH value of the mixture of colloidal solution with honey is about 4.9 and does not change over time. Also, it was observed that the stability of silver nanoparticles when honey is added does not change and that the particles remain stable over time.

The following examples show stable mixtures of alginate, honey and silver nanoparticles obtained by the same procedure, but with a change in the mass fraction of the components in the mixture.

Example 2.

Stability of the mixture: the pH value stabilizes at about 7 after 8 days, while the concentration of silver nanoparticles remains unchanged.

Example 3.

Stability of the mixture: the pH value stabilizes at about 5 after 8 days, while the concentration of silver nanoparticles decreases by about 40%.

Example 4.

Stability of the mixture: the pH value stabilizes at about 4.8 after 8 days, while the concentration of silver nanoparticles decreases by about 20%.

Example 5.

Stability of the mixture: the pH value stabilizes at about 5.6 after 8 days, while the concentration of silver nanoparticles decreases by about 13%.

Example 6.

Stability of the mixture: the pH value stabilizes at about 4.8 after 8 days, while the concentration of silver nanoparticles decreases by about 24%.

Example 7.

Stability of the mixture: the pH value stabilizes at about 4.4, and the concentration of nanoparticles decreases by about 25% after 9 days.

Example 8. Alginate microparticles with incorporated silver nanoparticles and honey

A mixture of alginate colloidal solution containing silver and honey nanoparticles in the amounts shown in Examples 1-7 is pushed through the tip of a 23G diameter flat-tip stainless steel needle (Small Parts, Inc. USA) using a liquid displacement infusion syringe pump (Racel, Scientific Instruments, Stamford, USA). A positively charged electrode was derived from a high voltage generator (Model 30R, Bertan Associates, Inc., New York, USA) and attached to the needle, while the grounded electrode was immersed in the gelling solution (1.5 wt.% calcium nitrate). An electrostatic voltage of 7.2 kV was applied, while the distance between the tip of the needle and the calcium nitrate solution was about 2.5 cm. The pressurized solution is detached from the tip of the needle under the influence of gravitational and electrostatic force in the form of a stream of tiny charged droplets and is collected in the gelling solution. Droplets in the calcium nitrate solution solidify in the form of microparticles due to the exchange of sodium and calcium ions. The microparticles are left in the gelling solution for another 30 min with stirring so that the gelling is complete. The microparticles obtained from the mixture containing 10% honey by electrostatic extrusion are spherical and uniform (diameter 740 ± 20 µm).

Example 9. Alginate microfibers with incorporated silver nanoparticles and honey

A mixture of alginate colloidal solution containing silver and honey nanoparticles in the amounts shown in Examples 1-7 is forced through a stainless steel flat tip needle (23 G) (Small Parts, Inc. USA) using a peristaltic pump (Berh Labor-Technik, Dusseldorf, Germany). The distance between the tip of the needle and the gelling solution of composition 6 wt. % calcium nitrate, is about 1 cm. A stream of silver-alginate colloidal solution with honey is collected in a calcium-nitrate solution, which results in the formation of microfibers. After formation, the microfibers are left to gel for another 2 hours, in order to ensure complete gelation of the alginate. Microfibers obtained by extrusion of a mixture containing an alginate colloidal solution with silver nanoparticles and honey in a ratio of 7:3 have a diameter of 420 ± 50 µm.

Example 10. Dry alginate microfibers with incorporated silver nanoparticles and honey

The microfibers obtained in the previous example are dried in air at room temperature, which preserves the silver nanoparticles in a percentage of 70-80%. The diameter of the dry fibers from the previous example is 170 ± 40 pm.

Example 11. Alginate plates with incorporated silver nanoparticles and honey

A mixture of colloidal solution of alginate with the content of nanoparticles of silver and honey in quantities

shown in examples 1-7 is poured into a mold. After 48 hours at room temperature, a dried film is obtained, which is then gelled by pouring calcium nitrate solution (6 wt.%). The film is left in the gelling solution overnight to allow it to gel completely. The obtained hydrogel is washed with distilled water.

Example 12. Dry alginate film with incorporated silver nanoparticles and honey

The nanocomposite hydrogel in the form of plates obtained in the manner described in example 11 is dried at room temperature to a constant mass. Dry hydrogels in the form of a film obtained from a mixture containing 50% honey are about 1 mm thick. The presence of silver nanoparticles in all obtained forms was confirmed by UV-Vis spectroscopy, using a UV spectrophotometer (3100 Mapada, Japan), while the concentration of silver nanoparticles was determined by atomic absorption spectrometry, using a Perkin Elmer3100 spectrometer (Perkin Elmer, USA). On the other hand, the concentration of honey was determined by liquid chromatography, using an ISC 3000 DP liquid chromatograph (Dionex, Sunnvale, USA).

In the following examples, mixtures of polyvinyl alcohol, silver nanoparticles and honey are described.

Example 13

Stability of the mixture: the pH value stabilizes at about 5.2 after 4 days, while the amount of silver nanoparticles increased by about 28% when honey was added to a concentration of 1.75 mM and remains stable.

Example 14

Stability of the mixture: the pH value stabilizes at about 6.1 after 3 days, while the amount of silver nanoparticles increased by about 27% at the moment when honey was added so that the final concentration of nanoparticles is 1.09 mM and remains stable.

Example 15

Stability of the mixture: the pH value stabilizes at about 5.06 after 3 days, while the amount of silver nanoparticles increased by about 32% at the moment when honey was added so that the final concentration of nanoparticles is 1.08 mM and remains stable.

Example 16

Stability of the mixture: the pH value stabilizes at about 4.46 after 4 days, while the amount of silver nanoparticles increased by about 50% at the moment when honey was added so that the final concentration of nanoparticles is 1.08 mM and remains stable.

Example 17

Stability of the mixture: the pH value stabilizes at about 4.26 after 4 days, while the amount of silver nanoparticles increased by about 78% at the moment when honey was added so that the final concentration of nanoparticles is 1.75 mM and remains stable.

From the mixtures described in examples 13-17, nanocomposite hydrogels in the form of spherical particles, microparticles, fibers and microfibers are obtained by extrusion in a similar way as described in examples 8-10 using solutions for chemical cross-linking, such as e.g. boric acid, orthophosphoric acid, glutaraldehyde, or formaldehyde, or a subcooled solution for physical crosslinking, such as oil at about -20°C, and in the form of plates and films in the manner described in examples 11 and 12, whereby chemical or physical gelation of PVA is further achieved, e.g. by pouring with gelling solution, subjecting to y-irradiation or freeze-thaw cycles.

For testing the nanocomposite according to this invention in a biological experiment, the mixture from example 2 was selected. % Ca(N03)2 x 2H20 so that fibers are obtained. The fibers are then stretched by winding onto spools to obtain microfibers (diameter < 1 mm) with incorporated silver nanoparticles and honey. In doing so, depending on the gelation time, the amount of solution and the diameter of the fibers, the degree of encapsulation of the various components of honey in the hydrogel is obtained up to 10% of the initial amount. The resulting microfibers were air-dried to a constant mass.

Burns on adult male mice (Mus musculus) were induced for 10 hours with an aluminum square probe (5x5 mm, weight 50 g) heated in boiling water at 100°C. The animals were randomly divided into three groups of at least 6 animals each: group 1 - the burns were treated every day with the mentioned hydrogels, group 2 - the burns were treated every day with a commercial dressing based on calcium-alginate and honey, and group 3 - the burns were not treated but only wrapped with sterile gauze every day, which served as a control.

Burns treated with the hydrogels described in this invention (group 1) were consistently the smallest in area compared to the other two groups and healed the fastest. Namely, the wounds in this group reached their maximum area on the 6th day after treatment, which was 43 ± 10 mm<2>, in the group treated with the commercial preparation (group 2) the maximum area of the wounds was significantly larger (59 + 4 mm<2>) and was reached on the 7th day after treatment, while the area of the wounds in control animals (group 3) increased until the 9th day after treatment, when it amounted to 64 ± 35 mm<2.> Also the degree erythrema was the smallest in group 1 and the healing time was the shortest (21 days), a slightly higher degree of erythrema was in the group treated with the commercial preparation (group 2), and the healing time was 22 days, while in the control group (group 3) the degree of erythrema was the highest, and the time required for wound healing was the longest (24 days).

The results indicate that the alginate-based hydrogel described in this invention with incorporated silver nanoparticles and honey reduced the degree of tissue damage, so that the wound surfaces were smaller, the degree of erythema was lower, and the wound healing occurred earlier, which compared to the commercial preparation based only on alginate and honey indicates the improved functionality achieved by the described invention.

Claims (9)

1. Polymer nanocomposites with incorporated silver and honey nanoparticles that include stable mixtures and hydrogels of polymers such as sodium alginate or polyvinyl alcohol with incorporated honey and silver nanoparticles. 2. Polymer nanocomposites according to claim 1, characterized in that the stable mixture includes 50-95 wt.% colloidal solution and 5-50 wt.% honey. 3. Polymer nanocomposites according to claim 1 and 2, characterized in that the colloidal solution includes 1.80-3.50% sodium alginate and 1-2 mM silver nanoparticles. 4. Polymer nanocomposites according to claim 1, characterized by a mixture comprising 5-30 wt.% of honey and 70-95 wt.% of a solution comprising 11.8-14.1% polyvinyl alcohol, 0.9-1.3 mM silver nanoparticles, 2.6-4.7 mM AgN03 and 0.02-6.6 mol/dm<3>NaOH. 5. Method for obtaining polymer nanocomposites with incorporated particles and honey, characterized by the fact that the mixture according to claims 2 and 3 is extruded into a gelling solution containing multivalent cations such as magnesium nitrate, barium nitrate and calcium nitrate, preferably calcium nitrate with a content of 1.5-6 wt.% calcium nitrate in order to obtain particles, microparticles, fibers and microfibers. 6. The method according to claim 5 for obtaining alginate nanocomposites in the form of a plate, characterized in that the mixture according to claims 2 and 3 is poured into a mold in which it is left to dry, and then poured with a gelling solution containing multivalent cations, preferably a solution with 6 wt.% calcium nitrate. 7. The method according to claim 6 for obtaining polymer nanocomposite in the form of a film, characterized in that the plate from claim 6 is dried at room temperature to a constant mass. 8. A method for obtaining polymer nanocomposites according to claim 4, characterized in that nanocomposite hydrogels in the form of spherical particles, microparticles, fibers and microfibers are obtained in the manner of claim 5 using solutions for chemical crosslinking such as solutions of boric acid, orthophosphoric acid, glutaraldehyde or formaldehyde or a cooled solution, such as oil at about -20°C, for physical crosslinking. 9. The method for obtaining polymer nanocomposites according to claims 1 and 4, characterized in that the plates and films are obtained by the method of claims 6 and 7, whereby chemical or physical cross-linking of PVA is carried out by pouring with a gelling solution, by the effect of v^sulfation^Tft^ and freezing-thawing.